Novel immunological composition against cystoisospora suis

Abstract

The present invention relates to a composition comprising a C. suis hapless protein 2 (HAP2), a C. suis oocyst wall protein 1 (OWP1) and a C. suis tyrosine-rich protein (TyRP), which can be administered to a pregnant sow ante partum, to protect its piglets against suckling piglet coccidiosis. The invention also relates to the use of said composition, the method of preparing said composition and a kit of parts comprising the composition.

Description

[0001] NOVEL IMMUNOLOGICAL COMPOSITION AGAINST CYSTOISOSPORA SUIS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to an immunological composition comprising a Cystoisospora suis hapless protein 2 (HAP2), a C. suis oocyst wall protein 1 (OWP1) and a C. suis tyrosine-rich protein (TyRP). The composition can be administered to a pregnant sow ante partum, to protect its piglets against suckling piglet coccidiosis. The invention also relates to the use of said composition, a method of preparing said composition and a kit of parts comprising the composition.

[0004] BACKGROUND OF THE INVENTION

[0005] Neonatal porcine cystoisosporosis (coccidiosis) is a disease caused by the protozoan endoparasite Cystoisospora suis (C. suis). Coccidiosis is characterized by self-limiting diarrhea and reduced weight gain in suckling piglets, mostly in the first three weeks of their life. Upon infection of a host’s intestinal tract by C. suis, symptoms such as internal hemorrhage, diarrhea, necroses, inflammations etc. may occur. Consequently, animals suffering from coccidiosis can be found to show loss of appetite, emaciation, fatigue, lethargy, and general weakness. Ultimately and particularly in the case of young animals infected with additional enteropathogens, e.g., Clostridium perfringens, coccidiosis is known to cause considerably increased mortality.

[0006] Piglet coccidiosis is a global problem with epidemiological data describing a high prevalence of 30-60% in European countries, China, and North America. An estimated 1 billion animals produced in industrialized pig production per year are put at risk.

[0007] US2010015182A1 discloses a vaccine comprising two strains of Coccidia species.

[0008] Due to the short incubation period in piglet coccidiosis, it is not possible to actively vaccinate newborn piglets as such an immunization would require either repeated action of the vaccine or repeated application beyond the period of disease and must also be considered limited in effect since piglets in their first weeks of life do not operate a fully functional immune system yet. The control of C. suis infection and disease in pig herds is therefore a major stake for breeders.

[0009] The development of effective control measures has so far only been based on the application of antiparasitic compounds, e.g., Toltrazuril, however, these compounds need to be applied to every piglet individually, and the recent emergence of antiparasitic resistance highlights the need for alternatives.

[0010] W02003099325A1 discloses an anti-protozoal vaccine composition comprising a viable protozoa produced by an inventive culture method.

[0011] The developmental stages of C. suis were only recently modelled in vitro in porcine intestinal cells (Feix et al., 2020) and in the absence of host cells (Feix et al.,

[0012] 2021 ), as well as characterized on the transcriptome level (Cruz-Bustos et al., 2022) and on the proteome level (Cruz-Bustos et al., 2023). C. suis’ life cycle consists of intracellular asexual multiplication followed by sexual development. Formed oocysts are excreted with feces, ingested by another animal, and infect its gut epithelium to re-start the life cycle.

[0013] In vitro, inhibition of sexual stage-specific proteins oocyst wall protein 1 (0WP1) or hapless protein 2 (HAP2) resulted in a reduced number of C. suis oocysts (Feix etal.,

[0014] 2022). In this study, chicken immunoglobulin IgY sera were assessed after the chicken were vaccinated with recombinant HAP2, 0WP1 , or Enkurin (Enk) that inhibit the development of C. suis sexual stages in vitro.

[0015] However, a C. suis vaccine has not been developed to date. The early time point of infection, within a few days after birth, occurs before effective immunization of piglets can be achieved. Thus, vaccination of piglets is not sufficient as means for protection.

[0016] An alternative strategy could be the immunization of pregnant sows. Superinfection of sows ante partum and following experimental infection of their piglets with C. suis oocysts led to a milder course of coccidiosis in suckling piglets (Schwarz et al., 2014), proving that maternal C. suis superinfection can provide protection for the piglets. Antibodies and immune cells produced by the sow are transferred with the colostrum to the piglets, providing systemic as well as intestinal immunity.

[0017] However, superinfection with large numbers of C. suis oocysts imposes safety problems and concerns in animal breeding. Thus, live superinfection of pregnant sows with C. suis oocysts is thus unsuitable for commercial purposes.

[0018] Palmieri et al. (2017) describes about 1170 potential C. suis vaccine antigens identified by screening of the C. suis proteome. W02008034121A2 discloses a vaccine comprising a protozoan HAP2 antigen.

[0019] US2005260224A1 discloses a Pid protein from Cystosiospora as an antigenic component for a vaccine.

[0020] WO201 1071404A1 discloses an immunogen from Fasciola hepatica. WO2016144933A1 discloses antibodies against Toxoplasma gondii oocysts.

[0021] Therefore, there is still a strong and yet unmet need for a vaccine against suckling piglet coccidiosis caused by C. suis.

[0022] SUMMARY OF THE INVENTION

[0023] It is the objective of the present invention to provide a vaccine against suckling piglet coccidiosis caused by C. suis.

[0024] The problem is solved by the present invention.

[0025] It has been shown by the inventors that a composition against Cystoisospora suis (C. suis) administered to a pregnant sow ante partum successfully protected its piglets against suckling piglet coccidiosis.

[0026] According to the invention there is provided a composition comprising a C. suis hapless protein 2 (HAP2), a C. suis oocyst wall protein 1 (0WP1) and a C. suis tyrosinerich protein (TyRP).

[0027] In an embodiment of the invention, the HAP2 comprises the sequence SEQ ID NO:1 , the 0WP1 comprises the sequence SEQ ID NO:2, and / or the TyRP comprises the sequence SEQ ID NO:3.

[0028] According to a further embodiment, the composition further comprises an adjuvant, specifically the adjuvant is an emulsion.

[0029] Provided in a further embodiment the composition further comprises a protein selected from the group consisting of sexual development-linked proteins of C. suis, specifically from an oocyst wall protein comprising the sequence SEQ ID NO:4, a toxoplasma gondii family a protein comprising any one of the sequences SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, a toxoplasma gondii family d protein comprising any one of the sequences SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11 , or a tyrosine-rich protein comprising the sequence SEQ ID NO:12, or SEQ ID NO:13.

[0030] According to a further embodiment, HAP2 is present in an amount in the range of 50 pg and 500 pg per dose, specifically 50 pg and 200 pg per dose, more specifically of 100 pg per dose.

[0031] In a further embodiment, 0WP1 is present in an amount in the range of 10 pg and 100 pg per dose, specifically 20 pg and 70 pg per dose, more specifically of 50 pg per dose. In a further embodiment, TyRP is present in an amount in the range of 10 pg and 100 pg per dose, specifically 20 pg and 70 pg per dose, more specifically of 50 pg per dose.

[0032] According to a further embodiment, the composition is a lyophilized powder, a frozen liquid, or a liquid.

[0033] In a further embodiment, the proteins are recombinant proteins.

[0034] According to a further embodiment, the composition is for use in vaccinating an animal.

[0035] In a further embodiment, the animal is porcine.

[0036] According to a further embodiment, the composition is for inducing protective anti- C. suis immunity in sows and / or protecting their piglets against piglet coccidiosis.

[0037] Provided in a further embodiment the composition is for use in vaccinating a pregnant sow or gilt ante partum, specifically, 6 weeks, 4 weeks, and / or 2 weeks ante partum.

[0038] In a further embodiment, the composition is for use in re-vaccinating a sow at each weaning of its piglets.

[0039] According to a further embodiment, the piglets resulting from a porcine vaccinated with said composition have reduced C. suis shedding and less diarrhea as compared to piglets born by an unvaccinated sow.

[0040] According to a further embodiment the composition is administered as an injection, specifically an intramuscular injection.

[0041] Provided in a further embodiment is a method for production of the composition, comprising the sequential steps: a. recombinant expression of HAP2, 0WP1 and TyRP proteins in bacterial host cells; b. harvesting the host cells; c. purification of each protein from the host cells; and d. mixing the proteins; e. addition of an adjuvant; and f. optionally, addition of a further protein.

[0042] Provided in a further embodiment is a kit for inducing an immune response against C. suis, comprising: a. said composition; b. instructions for use; and c. optionally, a solvent and / or an adjuvant.

[0043] FIGURES

[0044] Figure 1. Single antigen-binding affinities of antibodies in different samples in v7tro._Single HAP2, TyRP or 0WP1 antigen-coated plates were used for ELISA analysis to test binding affinities to antibodies IgG (Fig. 1A) or IgA (Fig. 1 B) in blood, colostrum, or milk samples from sows or blood samples from piglets of vaccinated sows. Antibody titers are shown as optical density (OD) at 450 nm. Error bars signify the standard deviation of three replicate measurements.

[0045] Figure 2. Serological response to vaccination in sows. IgG (Fig. 2A) or IgA (Fig. 2B) antibody levels against vaccine antigens in blood serum of vaccinated and control sows measured by ELISA are shown at the day of vaccination (DO), 42 days after vaccination (D42) and 72 days after vaccination (D72). Antibody titers are shown as optical density (OD) at 450 nm. The threshold for seropositivity was set at a dilution of 1 :800 for IgG and 1 :200 for IgA, as determined by previous ELISA analysis. Error bars signify the standard deviation of three replicates. Significances are indicated with *:p<0.05, **:p<0.01 , ***:p<0.001 between two values, ns: not significant.

[0046] Figure 3. Temporal changes in antibody concentrations in colostrum and milk of sows after parturition. Temporal changes of IgG (Fig. 3A) or IgA (Fig. 3B) antibody levels against vaccine antigens in colostrum and milk of vaccinated and control sows measured by ELISA are shown. Colostrum was collected immediately after parturition (W0), milk was collected 1 (W1 ), 2 (W2), or 3 (W3) weeks after parturition. Antibody titers are shown as optical density (OD) at 450 nm. Error bars signify the standard deviation of three replicates. Significances are indicated with **: p<0.01 , ***: p<0.001 between two values.

[0047] Figure 4. Serum antibody levels in piglets receiving antibodies through colostrum and milk.JgG (Fig. 4A) or IgA (Fig. 4B) antibody levels against vaccine antigens in blood samples of piglets from vaccinated and control sows measured by ELISA are shown for samples taken 1 day post-partum and 9 days post-partum. Antibody titers are shown as optical density (OD) at 450 nm. The threshold for seropositivity was set at a dilution of 1 :800 for IgG and 1 :200 for IgA. Error bars signify the standard deviation of following replicates: n=38 for piglets from vaccinated sows, n=31 for piglets from control sows. Significances are indicated with ***: p<0.001 between two values, ns: not significant.

[0048] Figure 5. Sum of diarrhea days of experimentally infected piglets._The number of days piglets experimentally infected with C. suis experienced diarrhea was determined by evaluation of fecal consistency. Statistical analysis was performed utilizing an ordinary one-way analysis of variance (ANOVA), with significance set at p < 0.0005. Error bars signify the standard deviation of following replicates: n=38 for piglets from vaccinated sows, n=31 for piglets from control sows. Significances are indicated with ***: p<0.001 between two groups.

[0049] Figure 6. Course of mean daily oocyst shedding in experimentally infected piglets. Mean oocysts per gram of feces determined daily is shown for piglets from vaccinated (n=38 piglets) and control (n=31 piglets) sows over a time-period of 16 sampling days.

[0050] Figure 7. Average daily excretion of oocysts in experimentally infected piglets. Mean oocysts per gram of feces as determined daily is depicted as an average over the whole sampling period of 16 sampling days for piglets from vaccinated (n=38 piglets) and control (n=31 piglets) sows. Significances are indicated with **: p<0.01 between two groups.

[0051] Figure 8. Superiority test of vaccination. Superiority-inferiority analysis of oocyst excretion where a margin of non-inferiority denoting the largest clinically acceptable difference between vaccination and non-vaccination was set at x<95%.

[0052] DETAILED DESCRIPTION

[0053] Unless indicated or defined otherwise, all terms used herein have their usual meaning in the art, which will be clear to the skilled person. Reference is for example made to the standard handbooks, such as Sambrook et al., "Molecular Cloning: A Laboratory Manual" (4th Ed.), Vols. 1 -3, Cold Spring Harbor Laboratory Press (2012); Krebs et al., "Lewin's Genes XI", Jones & Bartlett Learning, (2017), and Murphy & Weaver, "Janeway's Immunobiology" (9th Ed., or more recent editions), Taylor & Francis Inc, 2017.

[0054] The terms “comprise”, “contain”, “have” and “include” as used herein can be used synonymously and shall be understood as an open definition, allowing further members or parts or elements. “Consisting” is considered as a closest definition without further elements of the consisting definition feature. Thus “comprising” is broader and contains the “consisting” definition.

[0055] The term “about” as used herein refers to the same value or a value differing by + / - 5 % of the given value.

[0056] As used herein and in the claims, the singular form, for example “a”, “an” and “the” includes the plural, unless the context clearly dictates otherwise. The herein referred to “Cystoisospora suis” (C. suis) is a coccidian species. It is a protozoan parasite also referred to as Isospora suis. C. suis’ life cycle consists of intracellular asexual multiplication followed by sexual development. Formed oocysts are excreted with feces, ingested by another animal, and infect its gut epithelium to re-start the life cycle. Upon infection of a host’s intestinal tract by C. suis, symptoms such as internal hemorrhage, diarrhea, necroses, inflammations etc. may occur. Consequently, animals can be found to show loss of appetite, emaciation, fatigue, lethargy, and general weakness. This disease caused by C. suis is referred to as neonatal porcine cystoisosporosis (coccidiosis). In suckling piglets, coccidiosis is characterized by selflimiting diarrhea and reduced weight gain, mostly in the first three weeks of their life.

[0057] The present composition specifically can be an immunological composition.

[0058] A “immunological composition” as used herein refers to a composition comprising molecules, such as proteins or polypeptides, that are immunologically active in the sense that once administered to an animal, it can evoke an immune response of the humoral and / or cellular type directed against the molecule. As described herein, the immunological composition contains a C. suis hapless protein 2 (HAP2), a C. suis oocyst wall protein 1 (0WP1) and a C. suis tyrosine-rich protein (TyRP), and optionally an adjuvant.

[0059] The composition of the invention may also be a pharmaceutical preparation, further comprising one or more pharmaceutically or veterinary acceptable vehicles, diluents, or excipients.

[0060] An "immune response" to a composition or vaccine is the development of a cellular and / or antibody-mediated response to the composition described herein in the host, i.e. , the animal. Usually, an immune response includes, but is not limited to, one or more of the following effects: the production of antibodies, B cells, helper T cells, and / or cytotoxic T cells, directed specifically to the proteins included in the composition described herein.

[0061] In general, an immune response is generated to an antigen (also referred to as immunogen) through the interaction of the antigen with the cells of the immune system. Stimulation of an immune response can result from a direct or indirect response of a cell or component of the immune system to exposure to an immunogen. Immune responses may be broadly categorized into two categories: cell-mediated (Th1-type) and humoral (Th2-type, antibody-mediated) immune responses. Immune responses can be measured in many ways including stimulation of immunoglobulin (Ig) IgA, IgM, or IgG titer; activation, proliferation, or differentiation of cells of the immune system (e.g., B cells, T cells, dendritic cells, APCs, macrophages, NK cells, NKT cells, etc.); up- regulated or down-regulated expression of markers and cytokines; splenomegaly (including increased spleen cellularity); hyperplasia and mixed cellular infiltrates in various organs. Other responses, cells, and components of the immune system that can be assessed with respect to immune stimulation are known in the art.

[0062] Preferably, the host will display either a therapeutic or protective immunological response such that resistance to new infection will be enhanced and / or the clinical severity of the disease will be reduced. Such protection will be demonstrated by either a reduction or lack of symptoms and / or clinical disease signs normally displayed by an infected host, or a quicker recovery time of the infected host.

[0063] A “protein” is a macromolecule comprising one or more polypeptide chains. A protein may also comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but nonetheless may be present.

[0064] The term “amino acid” refers to one of the naturally occurring amino carboxylic acids of which proteins are comprised. Those amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, pyrrolysine, selenocysteine, serine, threonine, tryptophan, tyrosine, and valine. These 22 amino acids can be split into those that have neutral charges, positive charges, and negative charges:

[0065] The “neutral” amino acids are shown below along with their respective three-letter and single-letter code and polarity: alanine (Ala, A; nonpolar, neutral), asparagine (Asn, N; polar, neutral), cysteine (Cys, C; nonpolar, neutral), selenocysteine (Sec, U, nonpolar, neutral), glutamine (Gin, Q; polar, neutral), glycine (Gly, G; nonpolar, neutral), isoleucine (He, I; nonpolar, neutral), leucine (Leu, L; nonpolar, neutral), methionine (Met, M; nonpolar, neutral), phenylalanine (Phe, F; nonpolar, neutral), proline (Pro, P; nonpolar, neutral), serine (Ser, S; polar, neutral), threonine (Thr, T; polar, neutral), tryptophan (Trp, W; nonpolar, neutral), tyrosine (Tyr, Y; polar, neutral), valine (Vai, V; nonpolar, neutral), and histidine (His, H; polar, positive (10%) neutral (90%)). The “positively” charged amino acids are: arginine (Arg, R; polar, positive), lysine (Lys, K; polar, positive), and pyrrolysine (Pyl, O, positive).

[0066] The “negatively” charged amino acids are: aspartic acid (Asp, D; polar, negative), and glutamic acid (Glu, E; polar, negative).

[0067] Furthermore, the term “amino acid” includes both D- and L-amino acids (stereoisomers).

[0068] The term “polypeptide” as described herein refers to a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acids residues are commonly referred to as “peptides”.

[0069] The composition of the invention comprises three or more polypeptides:

[0070] A hapless protein 2 (HAP2), specifically comprising amino acid sequence SEQ ID NO: 1 , or an amino acid sequence having at least 98%, specifically at least 98.5%, 99%, or 99.5% identity to SEQ ID NO: 1 , or SEQ ID NO: 1 having 1 , 2, 3, 4, 5, or 6 point mutations. SEQ ID NO:1 can also be referred to as CSUI 000472. HAP2 from C. su / s is also referred to as CsHAP2.

[0071] SEQ ID NO: 1 is as follows:

[0072] MFNVTRDTID NRDKTFADVG NAGFSVSIKK TPVTISLPLE YIKDVPFDYR

[0073] EEIYEYSRWE AGRNPGKFCY ENTISKCSED DKLATHPSGK PLAWAHGRCC

[0074] WCSEVLAFTH INNMKRGNFR CNWFELVFAR SLWVTKSCPR TELPWYSMLR

[0075] INDGKQWTFS L EVE LR WWTP PRALQLITSK LFDECEAGKA EGTVPADRDC

[0076] VREKHERAGM TSRVYTLNFT SPEIFDRKRL

[0077] An oocyst wall protein 1 (0WP1), specifically comprising SEQ ID NO: 2, or an amino acid sequence having at least 98%, specifically at least 98.5%, 99%, or 99.5% identity to SEQ ID NO: 2, or SEQ ID NO: 2 having 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 point mutations. SEQ ID NO: 2 can also be referred to as CSUI 006207. 0WP1 from C. su / s is also referred to as CsOWPI .

[0078] SEQ ID NO: 2 is as follows:

[0079] MEPITSYEFY CVFLFKRSRP SFLAELTNMK LLVPWCAAA LSESFIHFSR

[0080] ANPGVPPVPS CPPGFTLEPR GCVRARQVPP VIRCPKKSVA SGNECVTKDF

[0081] AAGIEVCPEG FMEKNRKCRK VISVQPELGC KKGFALQAGG DCVRAGEDDV

[0082] ITRCPKHSKQ TSKGCITVQK VDVEYNCPEG LELRKNSCVG TQNIDAVPSC

[0083] PNGFLLESDM CVRKTATPPR VSCPKGYKAQ DNGCVLIEKF DLDVSCREGE

[0084] YDGKKHCRKT ALEPPAPHCP PGALLKDKEC VRVQEEPPRL VCDKGFTLQG NRCIREISVE AELSCPPEAQ LIKDKCAVLT SEKPALVCPD RFTVADGICK

[0085] AADVRPAELV CPKGYTADNR TGDCAKSAIV KPAISCERGE LRKGECVAVQ

[0086] TAPPELACMS GGMLTPEGCL FADTTPLLQR CPSDSKMDFN GKCSIVDWE

[0087] PEYICPPGFV PSGTKKATCE RFLTSPASMK CPAGFEPSGT KCMKTAWEP

[0088] IATCPEGLVF HQGTCIPYKS HGKKKA

[0089] A tyrosine-rich protein (TyRP), specifically comprising SEQ ID NO: 3, or an amino acid sequence having at least 98%, specifically at least 98.5%, 99%, or 99.5% identity to SEQ ID NO: 3, or SEQ ID NO: 3 having 1 , 2, 3, 4, 5, 6, or 7 point mutations. SEQ ID NO: 3 can also be referred to as CSUI 001473. TyRP from C. suis is also referred to as CsTyRP.

[0090] SEQ ID NO: 3 is as follows:

[0091] MEALRFSIVL LLLAAPIVAR CETSSQIVDS EDLMMEQEEM SIQEVHEMSE DMTSSPLRYL EEEESSEGMY IVPETFTPLR SLGKKNRAVY VAAPAKYVAP

[0092] AVHKKAAAPI QYVQAPLKYT PAISKKGRRL AAEMPVEEEM STISVDSELD

[0093] GDRDLHRTRG YYVAYTPYAY APYVHASPYC AMGSSCARYL NTEVDSEDVS

[0094] EGLIESAPER SMGRKSRAVY TAPMPKTYVP APAHKKAAAP VYRPATWQK

[0095] STPVHKSTPV TKKSPPWHK KSAPTPRSKV YQPAVTHTSS KYMPSVSKKG

[0096] RRLAEEAVSE EEATISEGET EQEERELKKR AAYYYPVYPV VAAPYCSKGT GCY

[0097] An oocyst wall protein which can also be referred to as CSUI_008806, comprising SEQ ID NO: 4, or an amino acid sequence having at least 98%, specifically at least 98.5%, 99% or 99.5% identity to SEQ ID NO: 4, or SEQ ID NO: 4 having 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 point mutations.

[0098] SEQ ID NO: 4 is as follows:

[0099] MEQRGFFLVI LCLAARAWA QDWPLIAGN EDWAAAEEC PSGYTLAAGV

[0100] CRKEVARKPL ALCPPRASYE NGECVTEREV KSVLTCSEGE RLIGDKCEVE

[0101] DVIKALASCP RDYTFTGTAC MRSQETRAVP RCDEGYKLSG DVCVRDVKTK

[0102] PDTYCPPGSR RSGDKCILVE SLPSHAACAR GYQLENGLCV KVDTVRADQK

[0103] CPHGFRMDHG VCKNWRLQP NAVCPSGYDF NGKECILSQL TEPTWKCEDG

[0104] YQLEGSTCVK RLEKAAKPEC PPKFDYKNGV CIRQTSVKPA SECPEGTVET

[0105] ANGKGCEAVH VADATLVCPN DYSVYNGQCV RRTTGGMHQE CEPGFKLTRE

[0106] GMCVRESSQK AEQRCPDGLE LVKDGYCVSQ DNDSPHYTCE QGELTPQGTC

[0107] VRVFTAETQF TCPRGFKLVE PNCVRQVQRT AVATCPDGSK MRGGSCIVLE

[0108] TFPPSHNCDD GYISDGYANC IKYETKNPKR TCPRSYKLFN TVCVKRGFRV A toxoplasma gondii family a protein which can also be referred to as CSUI 002027, comprising SEQ ID NO: 5, or an amino acid sequence having at least 98%, specifically at least 98.5%, 99%, or 99.5% identity to SEQ ID NO: 5, or SEQ ID NO: 5 having 1 , 2, 3, 4, or 5 point mutations. SEQ ID NO: 5 is as follows:

[0109] MLRVIVCLW FTGCVIQGRA EEETPTADFT EKIPKGGLPS TTQRSFWLKP

[0110] GQVLEWDEE GKADFEPVPA SGDGVAASTN SIFDPTKQGS LNAVAYAFKN

[0111] GACDFSTKIN YKDAFKKLDS YTSPLWKPET KQAVNAQLTN SVAKSYTFTN

[0112] PPXXXXXXFC VRFKTPKQEQ QSLRSVQEVG PAGGETAYLE VAIHSGAAGL

[0113] TAKILGFLAL TAAGIGKAIS L

[0114] A toxoplasma gondii family a protein which can also be referred to as CSUI 006655, comprising SEQ ID NO: 6, or an amino acid sequence having at least 98%, specifically at least 98%, 99%, or 99.5% identity to SEQ ID NO: 6, or SEQ ID NO: 6 having 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 ,15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, or 38 point mutations. SEQ ID NO: 6 is as follows:

[0115] MARQLGPRGV AFVALSLAVF CRLFVQLSVL AAPQGQPDYS IVIPIDGIPR

[0116] DDRYDFWLEG DDTFQIVDKS PGFSAIIEPP TFSQEAYAYS SPDCDLTKKI

[0117] EYKQEFSHAK EGHTFWKRDL ERDALDGRKY TFTMPPPEDL LDMVEFCLVI

[0118] TAPSKARDPD DPNWFRRRL QDIESFDATE HSLTWIHAG SQTLSTPELW

[0119] LCSPPRSLPD PPPGKEREVE DPLKRGPGSH GVLEWLIDAK NNLRHRPDPQ

[0120] LGRTIGGRVL SANDARLASS EAHKKASSSD TDEEERKRRK EWMALRRAHQ

[0121] LELRQQIRRR YEQIETEKSP PSQDGCRSDD SFSSQRGGRR GDLHQSSHRS

[0122] RAIGGGQPPR THHKGCGTSD GLGGGRGGHK EEDDGDGGAI PQDPTAMSLA

[0123] HFLKKNAQQL RDIHLRRHRR CRRRYSHESS SRQEDSDDMA VTRRSHQDRR

[0124] GGKKESNLNA VELSTDLERR RRHHLSLRGN SQKEHEVHKD SSPTYRGKLF

[0125] VDKGVGERQQ EDSWFSDSHF DDSSGDEAFV CRLSAPSLDS NDLKEEDQDE

[0126] RKKKKTSGEE SFEGPHGCYD EDDHYHHNSH GNSPSCTYNK KKCQEDFHSI

[0127] SPPLSPSKHT SHGVSSLDSY IRKGDGLDKG SEGEGGAIRA PRRQASMEDH

[0128] HRGSRLRPPL RENGGRGRGE GEAHQAPDET SHGQSEDSTS SKDERSISRW

[0129] SEESEGDKNF GRIPVSPLNP SYVSDGGSQT EIHRNSIDLH RQKCSPRPPP

[0130] SNGGVARRVS NSPTSSHSSC ISSSSSPPLV PIDGRSGGES EKEESPWYRD

[0131] GASHTSCPSS SFSSSSYDKR PSDAKKQIRH PFRHLCPNES IPGSTSTSQR

[0132] VDKSMKPFVS SQAKRGSFGS GGIAGCLVDD LAPQSPLPRG SNRKKREKKS LTGLFALDPR HLNLTSSSPT TSSGVYAPLS SPSHDGHAGG DGRISSSNSY

[0133] HPIPSSSSSL SPSRRAHAPL FPRISTTTSS STSQHFSRST PPCVSFPHPS

[0134] NQRTSTTSSS SSSSSSFFSP PSMSPPSPAH PSSLFYPCSS PSPSSSSPPP

[0135] SSTLTRHPSL IPTRTSDFTD PLGGAGNAGI GMGGEVFGED LSSSPGFSHL

[0136] GFASSSFPVS FPHNDQHLGN YFNHSFPLSP HTRRVSLPEF SRLKDQGGTS

[0137] HLHAACSSSN SPTNKKNPPY SLKDLPDSPG PQKLPYRPAS SKIRRDTQSP

[0138] DRRRGGNFHH RSHRERKKGR EHSQKRSSSK GARKDREERR DLTPQEISER

[0139] DSHRQEYHQH EYKNPYLSLH RGRDSYPSSS FYSPSPSPLV EISKNRRTAS

[0140] PDEDDRLNVT SSLSAPSRDF EKKTFIKGEE EEKEKEKESL SHEDEERERI

[0141] SRRRNAQSSR RQLLKSILEE TEREEEEENY GRRCSTKGED QGGREKKREK EKEKDGPLSS RLPGSSREKG LPPLFLSHRG IPGRGEEEER EREEDEDREE

[0142] EEEGEIKIRI RNYNSDDPDD CFSLEEEKER EEEKEEDEEK DQEREIRATP HHFSQQRGWL GGSEGRFLDK KKCHEDKPLL FLSPHITRVD EEPGRRRGRR HLSSFLSSDE GDSQERERTP ASYRFIQPRD SDEEEEEEEE EDVHNHESSS

[0143] SSSSPPPFPS SLPPQCWPR EEKGEREEEG EERTSVRLES KHPSCSIYDR

[0144] EHATDRMIRP LLKDSHGIPH GYNYPVLKSD VSSPSSSSSP SAIATRQRRL

[0145] LYLLKGRRGD GGDTFRSSSS ASSPSMSTGH HRPSPLGRRE GWKKQEEDED EEGRIHSLRE EDRSSSNDSA FCGWLMSPCS SIEGTGQVPP HPLFSSTASF

[0146] SSFPHSSSSS SSLPSFLRNT IHPLQVETTS TTPSSSLTTA SSLPSISSSS

[0147] SSAVATNNDR KRRDKGREGS GGMILAWSSP YEKNG

[0148] A toxoplasma gondii family a protein which can also be referred to as CSUI 010157, comprising SEQ ID NO: 7, or an amino acid sequence having at least 98%, specifically at least 98.5%, 99%, or 99.5% identity to SEQ ID NO: 7, or SEQ ID NO: 7 having 1 , 2, 3, 4, 5, or 6 point mutations.

[0149] SEQ ID NO: 7 is as follows:

[0150] MPQGARPAGQ QNEGLNSWY AVENGDCHPS KTLTYDEVFP GLGSSDTKTF

[0151] WVKTTSALES GSQNSFRADV GSTPVTNYVF TNPPADKLQG KQVSFCVRFH

[0152] SPASVTSTTT VITTAGSVAT QESTQKPVAT QQNPQQTGTE DKPTPSAKVE

[0153] VHQPSLEDLV KKDSPREGHK LVEDKEEPVN LLEKDGDKKI VHKMPGVYLS

[0154] KQSADHEGET PVGFDLTVII HSSASHVTVH TLSIFSVLAS SLLGILQGAP SV

[0155] A toxoplasma gondii family d protein which can also be referred to as CSUI 003908, comprising SEQ ID NO: 8, or an amino acid sequence having at least 98%, specifically at least 98.5%, 99%, or 99.5% identity to SEQ ID NO: 8, or SEQ ID NO:8 having 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or, 12 point mutations. SEQ ID NO: 8 is as follows:

[0156] MVMSKKEAPP PWKQEVCET VPYQVQATCY KDVTKKEAYS CPETLEKEVC

[0157] KNVPVQVPDT CYKQVSQQVA YSCPQTQQKT ECYDVPHTCT KTEYVSEAYE

[0158] CGKDVCKSVP VQVPNTCYKT VQEQQAYPCQ KVKPEQFCAE VPFTVAKTCY

[0159] KEDVKTVPYT CSKTAYKQQC RKVAFQVENT CYKTVMQAQQ YPCTKTAYED

[0160] SCSDVAVQVP DTCYKPVQQQ KPYKCKKTLT RQQCTKVPVE VPSTCTKTVM

[0161] TKEAYDCSKT EYKTECTEEV AQAPCVGKEC KLRQLKKQRV CRQVPFTTKN

[0162] VCYKEVPTSQ AYKCSKTEYT EQCQEVPYEV PDTCYETITV QQPYKCSKTE

[0163] YKQQCKKVPV EVPHTCTKQV PAQEAYPCPK TEYKDECQDV PVQVPDTCYK

[0164] QQPIKVPYSC PETKYQSKCE TKMVPFEDTC YKTVPVQQAY SCSKTVMEQQ CAFVSQTCHK KVPTQVQYSC PKKECKQVAF QVEKTCYKSV PTKEAYKCSK

[0165] TVFENQCATQ SYTVAKTCYK DHVKQQAYPC METKYRTQCK TKKAVAPAPA

[0166] PWLSKKGM

[0167] A toxoplasma gondii family d protein which can also be referred to as CSUI 004489, comprising SEQ ID NO: 9, or an amino acid sequence having at least 98%, specifically at least 98.5%, 99%, or 99.5% identity to SEQ ID NO: 9, or SEQ ID NO:9 havingl , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, or 13 point mutations.

[0168] SEQ ID NO: 9 is as follows:

[0169] MTLSWHVNA KKAEWHVEQ EICEQVETKV KSTCFKDVTT KEAYSCPWQ

[0170] EKEVCKNVAV QVPSTCYKEV TEKQAYSCPK TKTEKQCQDV VETCYKTEKQ

[0171] TREYPCEGKE TCTMVPVEVP DTCVRKVDQQ QAYACEKTKM EQVCAEVAVT

[0172] VEKTCHKEEM QTVPYPCTKT EYKQQCQQVA VPKAAAQTTC FKTVMKAQDY

[0173] PCSKTQYEQS CAQVPIEVKG KCHRMVPKEK PYKCKKTELK MKCKKANVKV

[0174] PSVCTKNVKV KEAYDCTQTE YKTECIVDVP CKEKKCKMRR LNEAANCQTV

[0175] PVQVPKTCYK EVDSVQEYEC EKKEQQEQCT EQEVEVNDTC YETVEVKEAY

[0176] ECIKVEYKEE CKKVAVEVPS TCTKEVPFQE AYPCPPVMEM QEKCEQVAAE

[0177] VQATCEKQVA VQVPYSCPAT EMKMQCSEQA VPYQDTCYKT VSVQEQYACT

[0178] KTTKQKQCHR EAKTCSEKVS VEVPYECTKK KCEKVDVQVD ETCYQKVKVQ

[0179] QPYACSETKY ENQCAVQTYS EPKTCYNDVL TKQAYPCYET KVETVCKPCA

[0180] AKVKITPEKV REPKQPKKAE AVWEQAPAV PVEAAPAVW HAPPPAPVLA

[0181] SKEPWPVAS KGGAVF

[0182] A toxoplasma gondii family d protein which can also be referred to as CSUI 004212, comprising SEQ ID NO: 10, or an amino acid sequence having at least 98%, specifically at least 98.5%, 99%, or 99.5% identity to SEQ ID NO: 10, or SEQ ID NO: 10 havingl , 2, 3, 4, 5, 6, or 7 point mutations.

[0183] SEQ ID NO: 10 is as follows:

[0184] MADCFQAQQV AVDCTEEVDT VQERQCPKTI KETVMHTVCE EPQYVPKKGF

[0185] HSRRLGPGKK GCRQVPQQIE RWMETCSEV VKVKVPKTCY QNQMVPVQST

[0186] CPQTEMEKVC FQVKQPVQKM CTREVPATED YSCDEWTET ECKKVPKQVQ

[0187] GVAYKEYEQK ESYKCEATEQ IKKCKMVPFF KASTCVAALM DVEAYECSVS

[0188] VMKEKCEQVA RTVPSTCVEE EWVTEQYECY EEGVERRCKT FPVYANAGTC

[0189] TATVKVTEEY PCTQPSLEQV CRMETSKVDN TCYEPVMEEQ KYTCFERHTR

[0190] QQCEKAPIPQ KKGGYHY

[0191] A toxoplasma gondii family d protein which can also be referred to as CSUI 009196, comprising SEQ ID NO: 11 , or an amino acid sequence having at least 98%, specifically at least 98.5%, 99%, or 99.5% identity to SEQ ID NO: 11 , or SEQ ID NO:11 having 1 , 2, 3, 4, 5, 6, 7, 8, or 9 point mutations.

[0192] SEQ ID NO: 11 is as follows:

[0193] MQKQPYSCTK TKPEQFCSEI PYTVNKVCHR EDSKLVNYPC TKTEYKQKCR KVPFQVDSTC YKTVMKPEEY KCSKTSYEQQ CTEVPVKVPD TCYRTVDQKK

[0194] SYKCKKTLTK NQCTKIPVEV PSTCSKTVMT KEAYDCSKTE YRTECSEEYE

[0195] QAPCMGKECK LRQLKKKRVC RQVPFTSKNV CYKKVPTQQT YPCTKTEYQQ

[0196] QCQEVPYEVP DTCYETVQVQ QPYKCHKTEY KQSCKKVPVQ VPDVCTRQVP

[0197] AKEPYSCPKT EYRQECEDVP VQVPDTCQKT VPTKVPYNCP ETKYRTKCET

[0198] KMVPVEETCY KTVPVKDAYK CTKTVTEKQC DYVSKTCSKQ VPMQTEYPCP

[0199] QKKCKQVPFE VDHTCYKNVP SQEPYKCSKT VMENQCSTQT YTVPKTCYED

[0200] QVKQQPYPCF ETKYRTQCKS KKGHHMPVSM ELSSPVFHSK GM

[0201] A tyrosine-rich protein which can also be referred to as CSUI_000190, comprising SEQ ID NO: 12, or an amino acid sequence having at least 98%, specifically at least 98.5%, 99%, or 99.5% identity to SEQ ID NO: 12, or SEQ ID NO: 12 havingl , 2, 3, 4, 5, or 6 point mutations.

[0202] SEQ ID NO: 12 is as follows:

[0203] MQLVPGVSW TAVGCFLVSL ASAEEAATEN TISPEIATAS AVEDGSPGYE ESTSERELTY GHGYYPGPVY QAPAAYYAPR PYYYFGRPRS YVRYLGAEKA QSQATEPEW ADPEEDLEGR SLGHRHHGPM YYGGYYGLVG YGPWYPPYG YFRYLKGMRR AAVASTADQD MPKRVAAARK MGHRRHGYYY APMSYYRPYY GYYSSPYGYV RYLRRAEGAG RGTVSDSASV QSEERQLSPH HRGYYGGPVF YGPGYYAPVP YGPYYGSPYI RYLREKPEAT WNESFEA

[0204] A tyrosine-rich protein which can also be referred to as CSUI_001475, comprising SEQ ID NO: 13, or an amino acid sequence having at least 98%, specifically at least 98%, 99%, or 99.5% identity to SEQ ID NO: 13, or, 1 , 2, 3, or 4 point mutations.

[0205] SEQ ID NO: 13 is as follows:

[0206] SKKWRRLSAL AETSQSEEVL SQAAEDTHDQ SFSQTANSTE ESESQAELLS

[0207] QAVEDTHDQS FSQTANSTEE SESQAETQTT EGEDDRDLGH RKSMYWPTV

[0208] ASWPVAHCM PTEPCYHGYA SSCMGSACHR YMGESEISSS ETSMGEESGE

[0209] ERSLGKKKQQ VWAAPVKKA APVKKAAPVM AVPTKKAAPV M

[0210] The composition according to the invention may comprise or consist essentially of one or more adjuvants. The term “adjuvant” refers to a substance, or a combination of substances, that is used to increase the efficacy or potency of drugs, specifically, a substance that increases the immune response to the composition described herein. Suitable adjuvants can be, but are not limited to (1 ) emulsions, specifically water-in-oil, oil-in-water, or water-in-oil-in-water emulsions. Specifically, emulsions made from mineral oil, non-mineral oil, or a composition of both, optionally, with surfactants. One commercially available emulsion-based adjuvant which is well known is Montanide™, specifically, Montanide™ ISA 201 water-in-oil adjuvant as used for the examples in the present invention. Adjuvants can further be, but are not limited to, (2) aluminum hydroxides or aluminum phosphates; (3) polymers of acrylic or methacrylic acid, maleic anhydride and alkenyl derivative polymers, (4) immunostimulating sequences (ISS), such as oligodeoxyribonucleotide sequences having one or more non-methylated CpG units, (5) cation lipids containing a quaternary ammonium salt, e.g., DDA, (6) cytokines, e.g., IFN-y, GM-CSF, or IL-4, (7) saponins, or (8) any combinations or mixtures thereof.

[0211] “Sexual development-linked proteins of C. suis as used herein refers to proteins whose coding transcripts have been found upregulated in sexually differentiated developmental stages (gamonts, gametes) or oocysts (oocyst wall composition or surface) as described in Cruz-Bustos et al., 2022.

[0212] The herein referred to “dose” refers to each administration of the composition. The dosevolume of the herein described composition for the target species, e.g. the dose volume of swine compositions, is generally in the range of about 0.1 to about 2.0 ml, specifically of about 0.1 to 1 .0 ml, specifically of 0.5 to 1 .0 ml, or specifically of 1 .0 to 2.0 ml. A dose of the composition specifically comprises a) a C. suis hapless protein 2 (HAP2) in the range of 50 pg and 500 pg, specifically 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 260, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 480, 480, 490, 500 pg; b) a C. suis oocyst wall protein 1 (0WP1) in the range of 10 pg and 100 pg, specifically 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 pg; and c) a C. suis tyrosine-rich protein (TyRP) in the range of 10 pg and 100 pg, specifically 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 pg.

[0213] HAP2 may be present in an amount between 50 pg and 500 pg per dose, specifically between 50 pg and 200 pg per dose, more specifically of 100 pg per dose.

[0214] The administrations of the composition are preferably carried out 1 to 6 weeks apart. Preferred time interval is 2 to 3 weeks, specifically 2 weeks. Specifically, a total 3 doses is given to the pregnant sow or gilt 6 weeks, and / or 4 weeks, and / or 2 weeks ante partum. It also refers to a re-vaccination of the sow after weaning of piglets.

[0215] Administration of the composition can be performed as an injection, specifically intramuscular, subcutaneous, or transcutaneous, or spray, intra-nasally, intra-ocularly, intra-tracheally, and / or orally, administration through inhalation.

[0216] The term “lyophilized” refers to substances processed by lyophilization, a low temperature dehydration process that involves freezing the product and lowering pressure, thereby removing the ice by sublimation. As used herein “lyophilized powder” refers to an composition processed by lyophilization. As used herein, a “lyophilized powder” can be dissolved in solvent, specifically water, buffer, or in an adjuvant.

[0217] The term “recombinant protein” refers to proteins which are formed by transfecting foreign genes into a host cell. Specifically, a host cell is a plant cell, an insect cell, a mammalian cell, a bacterial cell, or a yeast strain. Specifically, a host cell is selected from, but not limited to, Escherichia coli, Saccharomyces sp. Saccharomyces cerevisiae, Schizosaccharomyces sp. Schizosaccharomyces pombe, or Komagataella sp. Production of recombinant proteins can be in an expression system, specifically, expression of a recombinant protein in host cells, harvesting of host cells, purification of the recombinant protein, and mixing of recombinant proteins to obtain the composition, a process well-known to the person skilled in the art. The term “therapy” as referred to herein comprises administration of the composition as prophylaxis again C. su / s infection, specifically the composition is used as vaccine for vaccination.

[0218] The herein referred to “protection against disease” induced by the composition may be observed by reduced parasite, e.g. C. su / s shedding or reduced diarrhea in piglets of a vaccinated sow compared to piglets from an unvaccinated sow.

[0219] Due to vaccination with the composition described herein, the piglets have significantly fewer days of diarrhoea compared to the piglets from non-immunized mothers. Specifically, the piglets have at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% less days of diarrhoea compared to piglets from an unvaccinated sow.

[0220] The term “infectious” as used herein refers to a parasite, specifically C. su / s, that can invade and multiply in a host cell and causes a reaction of host tissue, specifically an immune response.

[0221] The term “antibody” refers to the secreted form of a B cell receptor. The term immunoglobulin (Ig) can refer to either the membrane-bound form or the secreted form of the B cell receptor. The terms antibody and immunoglobulin are herein used interchangeably.

[0222] One method to measure the presence of antibodies in liquid samples is the enzyme-linked immunosorbent assay (ELISA). The quantitative analysis of the ELISA is usually based on detection of intensity of transmitted light by spectrophotometry, which involves quantitation of transmission of some specific wavelength of light through the liquid, e.g., optical density (OD) measurements. As described in the examples herein, the ELISA was used to detect antibody levels in serum, colostrum, or milk.

[0223] The term “antigen” refers to proteins, peptides, polysaccharides (chains of simple sugars), lipids, or nucleic acids, which can bind to a specific antibody and may trigger an immune response. Specifically, the term “vaccine antigen” refers to proteins CsHAP2 (also referred to as HAP2), CsTyRP (also referred to as TyRP) and / or CsOWPI (also referred to as 0WP1), as well as the proteins listed herein. One type of a vaccine using said antigens is a subunit vaccine. Subunit-vaccine encompasses a vaccine that contains purified parts of the pathogen that are antigenic, or necessary to elicit a protective immune response. Subunit vaccine can be made from dissembled pathogen particles or recombinantly produced proteins. As used herein the term “pregnant sow” refers to a pregnant female porcine or a sow or gilt prior to breeding. The term “gilt” refers to a female porcine of up to a year old, which has not been bred previously. The herein referred to “vaccinated sow” refers to a pregnant gilt or sow which has received at least one dose of the composition described herein before farrowing. Specifically, the vaccinated sow will transfer immunity through maternal colostrum to its piglets upon birth. The transferred immunity may be in form of antibodies against antigens of the composition, or immune cells. The term “unvaccinated sow” refers to a pregnant gilt or sow which has not received said composition ante partum and thus will not transfer immunity to its piglets.

[0224] “Suckling piglets” refers to piglets which receive or have received colostrum from their mother, specifically, piglets of up to 5 weeks old. An alternative term for suckling piglets is “weaner”.

[0225] Herein provided is also a kit of parts for immunizing animals against coccidiosis, comprising the composition described herein in a container or dispenser capable of administering the composition, optionally a solvent and / or adjuvant, which can be already admixed with the composition or provided in a separate container, and instructions for use.

[0226] The present invention also encompasses the following embodiments:

[0227] 1. An immunological composition against Cystoisospora suis (C. suis), comprising: a. a C. suis hapless protein 2 (HAP2), b. a C. suis oocyst wall protein 1 (0WP1), and c. a C. suis tyrosine-rich protein (TyRP).

[0228] 2. The composition of embodiment 1 , wherein the HAP2 comprises the sequence SEQ ID NO:1 , the 0WP1 comprises the sequence SEQ ID NO:2, and / or the TyRP comprises the sequence SEQ ID NO:3.

[0229] 3. The composition of embodiment 1 or 2, further comprising an adjuvant, specifically the adjuvant is an emulsion.

[0230] 4. The composition of any one of embodiments 1 to 3, further comprising a protein selected from the group consisting of sexual development-linked proteins of C. suis, specifically from an oocyst wall protein comprising the sequence SEQ ID NO:4, a toxoplasma gondii family a protein comprising any one of the sequences SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, a toxoplasma gondii family d protein comprising any one of the sequences SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11, or a tyrosine-rich protein comprising the sequence SEQ ID NO:12, or SEQ ID NO:13.

[0231] 5. The composition of any one of embodiments 1 to 4, wherein HAP2 is present in an amount in the range of 50 pg and 500 pg per dose, specifically 50 pg and 200 pg per dose, more specifically of 100 pg per dose.

[0232] 6. The composition of any one of embodiments 1 to 5, wherein 0WP1 is present in an amount in the range of 10 pg and 100 pg per dose, specifically 20 pg and 70 pg per dose, more specifically of 50 pg per dose.

[0233] 7. The composition of any one of embodiments 1 to 6, wherein TyRP is present in an amount in the range of 10 pg and 100 pg per dose, specifically 20 pg and 70 pg per dose, more specifically of 50 pg per dose.

[0234] 8. The composition of any one of embodiments 1 to 7, wherein the composition is a lyophilized powder, a frozen liquid, or a liquid.

[0235] 9. The composition of any one of embodiments 1 to 8, wherein the proteins are recombinant proteins.

[0236] 10. The composition of any one of embodiments 1 to 9 for use in therapy.

[0237] 11. The composition of any one of embodiments 1 to 9 for use in vaccinating an animal.

[0238] 12. The composition for use of embodiment 11 , wherein the animal is porcine.

[0239] 13. The composition for use of embodiment 11 or 12 in inducing protective anti-C. su / s immunity in sows and / or protecting their piglets against piglet coccidiosis.

[0240] 14. The composition for use of any one of embodiments 11 to 13 in vaccinating a pregnant sow or gilt ante partum, specifically, 6 weeks, 4 weeks, and / or 2 weeks ante partum.

[0241] 15. The composition for use of any one of embodiments 11 to 14 in revaccinating a sow at each weaning of its piglets.

[0242] 16. The composition for use of any one of embodiments 11 to 15, wherein the piglets resulting from said porcine have reduced C. suis shedding and less diarrhea as compared to piglets born by an unvaccinated sow.

[0243] 17. The composition for use of any one of embodiments 11 to 16, wherein the composition is administered as an injection, specifically an intramuscular injection.

[0244] 18. A method for production of the composition of any one of embodiments 1 to 9, comprising the sequential steps: a. recombinant expression of HAP2, 0WP1 and / or TyRP proteins in bacterial host cells; b. harvesting the host cells; c. purification of each protein from the host cells; and d. mixing the proteins; e. addition of an adjuvant; and f. optionally, addition of a further protein.

[0245] 19. A kit for inducing an immune response against C. suis, comprising: a. the composition according to any one of embodiments 1 to 9; b. instructions for use; and c. optionally, a solvent and / or an adjuvant.

[0246] The examples described herein are illustrative of the present invention and are not intended to be limitations thereon. Different embodiments of the present invention have been described according to the present invention. Many modifications and variations may be made to the techniques described and illustrated herein without departing from scope of the invention.

[0247] EXAMPLES

[0248] Example 1

[0249] Binding affinity of antibodies to antigens in vitro

[0250] In a first example, the applicability of ELISA assay to test the binding affinity of the three vaccine antigens TyRP (comprising SEQ ID NO: 3), 0WP1 (comprising SEQ ID NO: 2) and HAP2 (comprising SEQ ID NO: 1) to antibodies was tested (Fig. 1). Fig. 1 shows data of an ELISA assay for single HAP2, TyRP, or 0WP1 antigen-coated plates to test binding affinities to antibodies IgG (Fig. 1A) or IgA (Fig. 1 B) present in blood, colostrum, or milk samples from vaccinated sows or blood samples from piglets of vaccinated sows. Antibody titers are shown as optical density (OD) at 450 nm. Error bars signify the standard deviation of three replicate measurements.

[0251] The three plates coated with single antigens showed a significant presence of immunoglobulin G (IgG) and immunoglobulin A (IgA) in both blood and colostrum, with a lower antibody concentration in milk. The three antigens displayed a similar trend in all cases. As a result, the ELISA analysis indicated differential binding affinities of antibodies to the three vaccine antigens across different sample types, i.e., blood, colostrum and milk samples from sows and blood samples from piglets of vaccinated sows.

[0252] To streamline the ELISA assay and enhance the comparative analysis of antibody concentrations across multiple antigens, multiplex antigen coating for ELISA plates was implemented. Applying multiple antigens to a single plate ensured uniformity in assay conditions across all antigens, which was vital for direct comparison. This uniformity minimized variations that could arise from running separate assays under potentially different conditions.

[0253] In vivo trial

[0254] Antibody responses to vaccination in sows

[0255] Then, the ELISA assay tested above was used to quantify IgG and IgA titers in the sera of vaccinated and unvaccinated sows (Fig. 2A for IgG and Fig. 2B for IgA). Fig. 2 shows IgG (Fig. 2A) or IgA (Fig. 2B) antibody levels against vaccine antigens in blood serum of vaccinated and control sows at the day of vaccination (DO), 42 days after vaccination (D42) and 72 days after vaccination (D72), measured by ELISA. Antibody titers are shown as optical density (OD) at 450 nm. The threshold for seropositivity was set at a dilution of 1 :800 for IgG and 1 :200 for IgA, as derived from previous ELISA analysis. Error bars signify the standard deviation of three replicates. Significances are indicated with *:p<0.05, **:p<0.01 , ***:p<0.001 between two values, ns: not significant.

[0256] As a result, a significant increase in IgG and IgA levels were measured after vaccination in the vaccinated cohort. Pre-vaccination blood taken 6 weeks ante-partum (indicated as DO) showed no significant IgG and IgA titers in either group. After vaccination, the vaccinated sows showed a significant increase in IgG levels, with the highest titers observed on the day before partum (indicated as D42). While IgA levels were also elevated in vaccinated sows post-vaccination, the increase was less pronounced than for IgG. In contrast, unvaccinated sows maintained baseline antibody levels throughout the whole study period. Measurements taken around five weeks postpartum showed that titers of both IgG and IgA in vaccinated sows remained elevated above pre-vaccination levels, suggesting a sustained immune response.

[0257] The concentrations of IgG and IgA in the colostrum and milk of sows were determined by ELISA at the day of birth and at three time points after parturition (Fig. 3A for IgG and Fig. 3B for IgA). Fig. 3 shows temporal changes of IgG (Fig. 3A) or IgA (Fig. 3B) antibody levels against vaccine antigens in colostrum and milk of vaccinated and control sows measured by ELISA. Colostrum was collected immediately after parturition (WO), milk was collected 1 (W1 ), 2 (W2), or 3 (W3) weeks after parturition. Antibody titers are shown as optical density (OD) at 450 nm. Error bars signify the standard deviation of three replicates. Significances are indicated with *:p<0.05, **:p<0.01 , ***:p<0.001 between two values, ns: not significant.

[0258] As a result, initial IgG and IgA concentrations in colostrum collected immediately after birth (indicated as WO) were markedly increased, reflecting the potential of colostral antibody transfer of immunoglobulins to neonates. These levels declined progressively in milk over the following weeks, with the lowest concentrations observed at the final sampling date (W3 post-partum). This trend is consistent with the natural decline in antibody levels as lactation progresses.

[0259] Successful colostral antibody transfer from vaccinated sows to their piglets

[0260] As a next step, colostral antibody transfer from sows to their piglet was investigated. A serological evaluation of the efficacy of passive immunization via colostrum and milk from vaccinated and unvaccinated sows to their piglets was evaluated using ELISA (Fig. 4A for IgG and Fig. B for IgA). Fig. 4 shows IgG (Fig. 4A) or IgA (Fig. 4B) antibody levels against vaccine antigens in blood samples of piglets from vaccinated and control sows measured by ELISA. Samples were taken 1 day postpartum and 9 days post-partum. Antibody titers are shown as optical density (OD) at 450 nm. The threshold for seropositivity was set at a dilution of 1 :800 for IgG and 1 :200 for IgA. Error bars signify the standard deviation of following replicates: n=38 for piglets from vaccinated sows, n=31 for piglets from control sows. Significances are indicated with *: p<0.05, **: p<0.01 , ***: p<0.001 between two values, ns: not significant.

[0261] As a result, blood samples from piglets from vaccinated sows contained specific IgG and IgA while sample from piglets from unvaccinated sows did not. Over time, these antibody levels declined as expected due to the natural decline in antibody concentration in the sow's milk and the breakdown of immunoglobulins in the piglets. The control group had consistently lower immunoglobulin levels, illustrating the effect of maternal vaccination on passive transfer of immunity.

[0262] Taken together, the presence of elevated antibody levels in blood and colostrum of vaccinated sows compared to unvaccinated sows demonstrated the effect of vaccination on antibody production, and initial antibody titers in piglets show transfer of these specific antibodies after birth. Colostral antibody titers were still elevated compared to the control groups at the age of three weeks.

[0263] Clinical and parasitological parameters

[0264] Diarrhea is a hallmark of suckling piglet coccidiosis, and the impairment of intestinal function leads to loss of fluids and nutrients and consequently stunted growth. Differences in the number of days piglets showed diarrhea was assessed for piglets born to sows of the vaccinated and control groups (Fig. 5). Fig. 5 shows an analysis of the number of days piglets experimentally infected with C. suis experienced diarrhea. Occurrence of diarrhea was determined by evaluation of fecal consistency. Statistical analysis was performed utilizing an ordinary one-way analysis of variance (ANOVA), with significance set at p < 0.0005. Error bars signify the standard deviation of following replicates: n=38 for piglets from vaccinated sows, n=31 for piglets from control sows.

[0265] As a result, the piglets from mothers of the vaccinated group exhibited significantly fewer days of diarrhea compared to piglets from non-immunized mothers. Instances of diarrhea were observed in some vaccinated piglets over the trial period; however, diarrhea can occur in piglets due to reasons other than coccidiosis, and it was not expected that vaccinated animals were free of diarrhea. Consequently, a significant reduction in diarrhea indicated an effect of vaccination on parasite development and consequently a reduced destruction of the intestinal lining.

[0266] Upon infection with C. suis, oocysts are excreted into the environment. Daily oocyst shedding was examined over a time period of around 2 weeks and compared between experimentally infected piglets from vaccinated sows and piglets from nonvaccinated sows (Fig. 6) Fig. 6 shows mean oocysts per gram of feces determined daily for experimentally infected piglets from vaccinated (n=38 piglets) and control (n=31 piglets) sows over a time-period of 16 sampling days.

[0267] As a result, for piglets of the control group, oocysts shedding was observed as two distinct peaks over the two-week period, during which the oocyst counts per gram of feces (OpG) were approximately four times higher in the control group compared to the vaccinated group. During the second peak at around sampling day 12, the OpG substantially increased to around 40,000 oocysts per gram of feces for the control group. Infected piglets from vaccinated mothers did not shed oocysts to this extent, with only one peak occurring around sampling day 6, where up to around 10,000 oocysts per gram of feces were measured. The area under the curve (AUC) for the OpG was similarly reduced in the piglet group coming from vaccinated sows (AUC = 9.02) compared to the control group (AUC = 90.08).

[0268] Taken together, the environmental oocyst contamination of the farrowing area of piglets born to vaccinated sows was shown to be significantly reduced.

[0269] Next, the average excretion of oocysts during daily sampling was assessed for piglets from vaccinated and unvaccinated sows (Fig. 7). Fig 7 shows mean oocysts per gram of feces as determined daily, depicted as an average over the whole sampling period of 16 sampling days for piglets from vaccinated (n=38 piglets) and control (n=31 piglets) sows.

[0270] As a result, the analysis revealed a significant (p<0.05) reduction in the total OpG in piglets born to vaccinated sows compared to those in the control group. Additionally, the variation within the vaccinated group exhibited a narrower distribution in contrast to the control group. Overall, the vaccinated group demonstrated a four-fold decrease in oocyst shedding compared to the control group.

[0271] Taken together, in addition to the shortened excretion period, the average amount of oocyst excretion was reduced in infected piglets from vaccinated sows.

[0272] To evaluate the impact of vaccinating sows on the infection of piglets with C. suis and their overall health during the course of infection, as a next step, it was determined whether the vaccination protocol employed would yield outcomes that were not significantly worse than the existing conditions, namely, the absence of any treatment. To this end, it was hypothesized that if the disparity in outcomes between the two treatments fell within a predetermined threshold, it was deduced that the vaccine was non-inferior to no treatment in terms of oocyst excretion (Fig. 8). Fig. 8 shows a superiority test where a margin of non-inferiority denoting the largest clinically acceptable difference between oocyst excretion following vaccination and nonvaccination was set at x<95%.

[0273] As a result, refraining from vaccinating the sows was determined to markedly exacerbate the existing conditions experienced by the piglets. Conclusion and industrial application

[0274] Vaccination of sows ante partum with an immunological composition comprising three recombinant C. su / s-specific proteins CsTyRP, CsHAP2 and CsOWPI , all related to sexual parasite development, induced production of antibodies IgG and IgA that were excreted with sows’ colostrum and milk. These antibodies could consequently also be detected in the blood of sows’ offspring in the first three weeks of life.

[0275] After experimental infection of piglets, infection took a clinically milder course, e.g., less diarrhea with reduced oocyst excretion determined as fewer days of excretion and lower OpG values in piglets from vaccinated sows compared to the control groups.

[0276] Taken together, the present invention discloses an immunological composition to vaccinate pregnant sows to prevent piglets from suckling piglet coccidiosis caused by C. suis. Piglet coccidiosis is a global problem with epidemiological data describing a high prevalence of 30-60 % in European countries, China, and North America, putting an estimated 1 billion animals produced in industrialized pig production per year at risk. To this date, no vaccine against C. suis exists, and control is limited to the use of the pesticide Toltrazuril, which must be applied to every single piglet and displays environmental toxicity. The vaccine disclosed in the present invention allows for protection of piglets at industrial scale through maternal vaccination.

[0277] Methods

[0278] Antigen production

[0279] Recombinant proteins for the immunological composition were produced in E. coli. Briefly, antigen was produced using a Champion pET151 Directional TOPO® Expression Kit for the cloning and the expression of recombinant proteins with N- terminal V5-6xHis tags following the manufacturer’s instructions. Coding sequences of the hypothetical gene CsTyRP (SEQ ID NO:14, also referred to as CSUI 001473), and the putative CsHAP2 (SEQ ID NO: 15, CSUI_000472) and CsOWPI (SEQ ID NO: 16, CSUI_006207) were amplified by PCR from C. suis cDNA using a Q5 high Fidelity® DNA Polymerase according to manufacturer’s instructions. Sequences are listed in Table 1. The gene-specific primers used for amplification and subsequent cloning into Champion pET151 Directional TOPO® are listed in Table 2. After verification of the correct cloning in One Shot® TOP10 E. coli and confirmation of the reading frames, plasmids with the correct inserts were used to transform BL21 Star® (DE3) One Shot® chemically competent E. coli.

[0280] _

[0281] Table 1. Coding gene sequences for hypothetical gene CsTyRP, and putative genes CsHAP2 and CsOWPI.

[0282] Table 2. Gene-specific primers used for amplification and subsequent cloning of vaccine antigen genes.

[0283] A detailed protocol on the cloning, production and purification of the proteins and the exact amount and dose processed is described below: The first section describes cloning and expression of the recombinant proteins, and the second section describes the production of recombinant proteins.

[0284] Section 1 : CLONING OF THE RECOMBINANT VACCINE ANTIGENS Equipment and materials used for cloning of the recombinant vaccine antigens are shown in Table 3, reagents and solutions used for cloning of the recombinant vaccine antigens are shown in Table 4. Table 3. Equipment and materials used for cloning of the recombinant vaccine antigens.

[0285] Table 4. Reagents and solutions used for cloning of the recombinant vaccine antigens.

[0286] Producing blunt-end PCR products

[0287] A 50 pl PCR reaction was set up (Table 5). PCR tubes were transferred to a PCR machine and thermocycling was performed as described in Table 6.

[0288] Table 5: PCR reaction set-up.

[0289] Agarose gel electrophoresis was used to validate the quality and quantity of the PCR product. TOPO Cloning Reaction

[0290] As a next step, the TOPO® cloning reaction was set up (Table 7). For optimal results, a 0.5:1 to 2:1 molar ratio of PCR product: TOPO® vector was used. The reaction mix was mixed gently and incubated for 5 minutes at room temperature. Then, the reaction mix was placed on ice to proceed to transform One Shot® TOP10 chemically competent E. coli.

[0291] Table 7. Reaction mixture for TOPO® cloning reaction.

[0292] Transform TOP10 Chemically competent E.coli 3 pl of the TOPO® Cloning reaction were added into a vial of One Shot® TOP10 chemically competent E. coli and were mixed gently. Then, the reaction mixture was incubated on ice for 5 to 30 minutes. The cells were heat-shocked for 30 seconds at 42°C without shaking. Then, the tube was immediately transferred to ice. 250 pl of room temperature S.O.C. medium was added and the mixture was incubated at 37°C for 1 hour with shaking. 100-200 pl of bacterial culture was spread on a prewarmed selective plate and incubated overnight at 37°C.

[0293] The next day, positive clones from overnight culture were analyzed: for each sample, 48 pl of PCR SuperMix High Fidelity were aliquoted into a 0.5 ml microcentrifuge tube. 1 pl each of the T7 forward and reverse PCR primer was added. 5 colonies were picked and resuspended individually in 50 pl of the PCR cocktail. PCR tubes were transferred to a PCR machine and thermocycling was performed as described in Table 6.

[0294] Agarose gel electrophoresis was used for visualization, and 5 positive colonies were picked and cultured overnight in LB medium containing the appropriate antibiotic. Plasmid DNA was isolated and sent for sequencing with T7 primers to confirm the correct of the reading frames.

[0295] Expression of PCR product

[0296] One vial of BL21 Star™(DE3) One Shot® cells per transformation was thawed on ice. 5-10 ng plasmid DNA in a 1 to 5 pl volume was added into each vial of BL21Star™(DE3) One Shot® cells, mixed by stirring gently with the pipette tip, and incubated on ice for 30 minutes. The cells were heat-shocked for 30 seconds at 42°C without shaking, then the tubes were immediately transferred to ice. 250 pl of room temperature S.O.C. medium was added. The tube was tightly closed, and tapde on its side for better aeration, and incubated at 37°C for 30 minutes with shaking (200 rpm).

[0297] The entire transformation reaction was added to 10 ml of LB containing the appropriate antibiotic (and 1 % glucose, if desired) and grown overnight at 37°C with shaking.

[0298] 10 ml of LB containing the appropriate antibiotic was inoculated with 500 pl of the overnight culture, and grown for two hours at 37°C with shaking. The OD600 should be about 0.5-0.8 (midlog).

[0299] The culture was split into two 5 ml cultures. IPTG was added to a final concentration of 1 mM to one of the cultures, to obtain one induced, and one uninduced culture. A 500-pl aliquot was removed from each culture, centrifuged at maximum speed in a microcentrifuge for 30 seconds, and the supernatant was aspirated.

[0300] The cell pellets were frozen at -20°C. These samples were referred to as the zero time point samples. Incubation of the cultures was continued at 37°C with shaking. Every 4 hours, time points for each culture were taken. For each time point, 500 pl from the induced and uninduced cultures were removed. The samples were thawed, and each cell pellet was resuspended in 80 pl of 1X SDS-PAGE sample buffer. Samples were boiled for 5 minutes and centrifuged briefly. 5-10 pl of each sample was loaded on an SDS-PAGE gel and electrophoresis was performed.

[0301] Section 2: PRODUCTION OF THE RECOMBINANT VACCINE ANTIGENS

[0302] Equipment and materials used for recombinant vaccine antigen production are shown in Table 8, reagents and solutions used for recombinant vaccine antigen production_are shown in Table 9. Table 10 describes used buffers for purification under denaturing conditions.

[0303] Table 8. Equipment and materials used for recombinant vaccine antigen production.

[0304] Table 9. Reagents and solutions used for recombinant vaccine antigen production.

[0305] Table 10. Buffers for purification under denaturing conditions.

[0306] Growth and induction a 500 ml bacterial culture

[0307] 15 ml of LB containing the appropriate antibiotic (100 pg / ml ampicillin; Sigma- Aldrich, St. Louis, Missouri, USA) was inoculated with the culture of the transformation reaction, and grown overnight at 37°C with shaking (180 rpm) to OD600 = 1-2.

[0308] The next day, 500 ml of LB containing the appropriate antibiotic (ampicillin, 100 pg / ml, Sigma-Aldrich, St. Louis, Missouri, USA) was inoculated with 5 ml of the overnight culture, and the culture was grown at 37°C with shaking (180 rpm) to an OD600 = ~0.5 (1 hours).

[0309] 1 mM IPTG (500pl stock 1 M), (Sigma-Aldrich, St. Louis, Missouri, USA) was added to induce expression, and cells were grown at 37°C with shaking for 4 hours. Then, the cells were harvested by centrifugation at 4500rpm for 15 minutes at +20°C. Cells were stored at -20°C for future use if not immediately purified.

[0310] Lysis

[0311] The cell pellet was thawed for 15 min on ice and resuspended in buffer B at 40 ml per 500ml of culture (for two purification column steps). Cells were stirred for 15-60 min at room temperature or lysed by gentle vortexing. Lysate was centrifuged at 15,000 x g for 20 min at 4°C to pellet the cellular debris. Supernatant, the cleared lysate containing the proteins, was saved. Lysates were analysed by SDS-PAGE (12.5%) followed by staining with Coomassie blue.

[0312] Purification of 6xHis-tagged proteins using Ni-NTA Superflow under denaturing conditions

[0313] The purification column (His GraviTrap®, GE Healthcare, Chicago, Illinois USA) was assembled according to the manufacturer’s instructions. The top adapter of the column was removed, and the bottom outlet was capped. A 50% Ni-NTA Superflow slurry was thoroughly resuspended and poured into the column. Note: The column and bed size depends on the amount of 6xHis-tagged protein to be purified. Generally, the binding capacity of Ni-NTA Superflow is 5-10 mg protein per ml resin.

[0314] The resin was allowed to settle, and the column was equilibrated with 50 ml of buffer B. The lysate was applied to the column, which was washed with buffer B until the A280 was below 0.01 .

[0315] A wash step with buffer C (20 ml) and D (10 ml) followed, checked with Bradford assay. The protein was eluted with buffer E (5-10 ml), and the concentration of purified protein was determined by Bradford assay using bovine serum albumin (BSA) as a standard. The confirmation and purity of the recombinant protein was analyzed by SDS- PAGE. Purified protein was concentrated using amicon tubes.

[0316] Analyzing samples by SDS-PAGE

[0317] Each protein was thawed and mixed together at equivalent amounts of supernatant and 2X SDS-PAGE sample buffer, and boiled for 5 minutes. 10 pl of the supernatant sample was loaded and a SDS-PAGE gel was run at 200V for 1 h. The polyacrylamide gel was stained with Coomassie blue and looked for a band of increasing intensity in the expected size range for the recombinant protein.

[0318] Dialysis

[0319] The samples were dialyzed against PBS 6 M urea, 4 M urea, 2 M urea and final PBS using the cassettes Slide-A-Lyzer™, 3.5K MWCO, 12 mL (ThermoFischer) as described as follows:

[0320] A syringe needle was inserted through the gasket via one of the corner ports. The sample was injected and the excess air was withdrawn and the syringe was removed. A float buoy was attached and dialysis was started. The cassette+buoy was introduced into 1 liter of PBS-buffer B 6M (Mix 0.75ml of Buffer B with 0.25ml of PBS).

[0321] The buffer was exchanged with PBS-buffer B 4M (Mix 0. 5ml of Buffer B with 0. 5 ml of PBS), then the buffer was exchanged with PBS-buffer B 2M (Mix 0.25ml of Buffer B with 0.75 ml of PBS).

[0322] Buffer was exchanged with PBS every 3h, then kept in PBS overnight (16 h).

[0323] An empty syringe needle was inserted at a second corner port. Air was injected to expand the cassette chamber, and then the dialyzed sample was withdrawn. The concentration of purified protein was determined by Bradford assay using bovine serum albumin (BSA) (Sigma) as a standard. The confirmation and purity of the recombinant protein was analyzed by SDS-PAGE.

[0324] Before use for vaccination, each protein was thawed at room temperature just prior mixing with the adjuvant Montanide as described in the next section.

[0325] Animals experiments in vaccine trial

[0326] Six sows were enrolled in the trial. Per injection, doses of 100 pg of HAP2 and 50 pg of each 0WP1 and TyRP were mixed with adjuvant (Montanide™ ISA 201 VG N ST, applied according to the manufacturer’s instructions). Each sow of the vaccination group received three doses starting six weeks ante partum in two week-intervals; control sows received adjuvant only in the same volume in parallel.

[0327] Piglets from these sows were enrolled when they had at least 900 g of body weight on the day of birth and were orally infected with 1 ,000 sporulated oocysts of C. suis (strain Wien-I) on the 3rdday of life.

[0328] Feces sampling and analysis

[0329] Individual fecal samples were systematically collected daily from day 5 to 16 after infection to assess fecal consistency and oocyst excretion. Fecal consistency was promptly evaluated upon sampling and categorized according to the following scale: fecal score (FS) 1 for normal, FS 2 for pasty, FS 3 for semi-liquid, and FS 4 for liquid consistency, with FS 3 and FS 4 indicative of diarrhea. Initial screening for oocysts was conducted through autofluorescence (AF) detection under UV light, with a sensitivity of approximately 10 oocysts per gram (OpG). In positive samples, oocyst excretion was quantitatively determined using a modified McMaster technique as described in Joachim, Ruttkowski and Sperling, 2018.

[0330] Statistical evaluation

[0331] The statistical evaluation encompassed three control groups (=litters) and three vaccinated groups (=litters), all subjected to normalization procedures. In total, 38 piglets from 3 vaccinated sows and 31 piglets from 3 unvaccinated (control) sows were included in the evaluation of fecal samples and antibody titers. Only piglets for which samples were available on all sampling days were included in the statistical analysis. Differences between the vaccinated and control groups were assessed utilizing an ordinary one-way analysis of variance (ANOVA), with significance set at p < 0.0005 unless indicated differently.

[0332] Antibody measurements and analysis To evaluate antibody formation and transfer, sows’ blood, colostrum and milk, and piglets’ blood was collected and subjected to analysis by ELISA. For this, plates were coated with the three recombinant antigens according to standard methods (50 ng / well), and HRP-conjugated secondary anti-pig antibodies against porcine IgG and IgA were used for detection. Samples were diluted 1 :100 to 1 :6,400 and analyzed in duplicates. Optical densities were analyzed and calculated in relation to the buffer control after determining test quality criteria with previously characterized pig sera.

[0333] REFERENCES

[0334] Cruz-Bustos, T., Feix, A.S., Lyrakis, M., Dolezal, M., Ruttkowski, B., Joachim, A. (2022): The transcriptome from asexual to sexual in vitro development of Cystoisospora suis (Apicomplexa: Coccidia). Sci. Rep. 12, 5972.

[0335] Cruz-Bustos, T., Dolezal, M., Feix, A.S., Ruttkowski, B., Hummel, K., Razzazi- Fazeli, E., Joachim, A. (2023): Unravelling the developmental biology of Cystoisospora suis, a model for comparative coccidian parasite studies. Frontiers Cell. Inf. Microbiol. 13: 1271731.

[0336] Feix, A.S., Cruz-Bustos, T., Ruttkowski, B., Joachim, A. (2020): Characterization of Cystoisospora suis sexual stages in vitro. Parasit. Vectors 13(1); 143.

[0337] Feix A.S., Cruz-Bustos T., Ruttkowski B., Mbtz M., Rumenapf T., Joachim A. (2021): Progression of asexual to sexual stages of Cystoisospora suis in a host cell-free environment as a model for Coccidia. Parasitology 148 (12), 1475-1481.

[0338] Feix, A.S., Cruz-Bustos, T., Ruttkowski, B., Joachim, A. (2022): Inhibition of sexual stage-specific proteins results in reduced numbers of sexual stages and oocysts of Cystoisospora suis (Apicomplexa: Coccidia) in vitro. Int J Parasitol. 2022;52(13- 14):829-841.

[0339] Palmieri, N., Shrestha, A., Ruttkowski, B., Beck, T., Vogl, C., Tomley, F., Blake, D.P., Joachim, A. (2017): The genome of the protozoan parasite Cystoisospora suis and a reverse vaccinology approach to identify vaccine candidates. Int J Parasitol. 2017;47(4): 189-202.

[0340] Ruttkowski, J.A., Sperling, C. D. (2018): Detection of Cystoisospora suis in faeces of suckling piglets - when and how? A comparison of methods. Porcine Health Manag. 2018 Sep 19;4:20.

[0341] Schwarz, L., Worliczek, H.L., Winkler, M., Joachim, A. (2014): Superinfection of sows with Cystoisospora suis ante partum leads to a milder course of cystoisosporosis in suckling piglets. Vet. Parasitol. 204, 158-168.

Claims

CLAIMS1. A composition against Cystoisospora suis (C. suis), comprising: a. a C. suis hapless protein 2 (HAP2), b. a C. suis oocyst wall protein 1 (0WP1), and c. a C. suis tyrosine-rich protein (TyRP).

2. The composition of claim 1 , wherein the HAP2 comprises the sequence SEQ ID NO:1 , the 0WP1 comprises the sequence SEQ ID NO:2, and / or the TyRP comprises the sequence SEQ ID NO:3.

3. The composition of claim 1 or 2, further comprising an adjuvant, specifically the adjuvant is an emulsion.

4. The composition of any one of claims 1 to 3, further comprising a protein selected from the group consisting of sexual development-linked proteins of C. suis, specifically from an oocyst wall protein comprising the sequence SEQ ID NO:4, a toxoplasma gondii family a protein comprising any one of the sequences SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, a toxoplasma gondii family d protein comprising any one of the sequences SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11 , or a tyrosine-rich protein comprising the sequence SEQ ID NO:12, or SEQ ID NO:13.

5. The composition of any one of claims 1 to 4, wherein HAP2 is present in an amount in the range of 50 pg and 500 pg per dose, specifically 50 pg and 200 pg per dose, more specifically of 100 pg per dose.

6. The composition of any one of claims 1 to 5, wherein 0WP1 is present in an amount in the range of 10 pg and 100 pg per dose, specifically 20 pg and 70 pg per dose, more specifically of 50 pg per dose.

7. The composition of any one of claims 1 to 6, wherein TyRP is present in an amount in the range of 10 pg and 100 pg per dose, specifically 20 pg and 70 pg per dose, more specifically of 50 pg per dose.

8. The composition of any one of claims 1 to 7, wherein the composition is a lyophilized powder, a frozen liquid, or a liquid.

9. The composition of any one of claims 1 to 8, wherein the proteins are recombinant proteins.

10. The composition of any one of claims 1 to 9 for use in therapy.

11. The composition of any one of claims 1 to 9 for use in vaccinating an animal.

12. The composition for use of claim 11 , wherein the animal is porcine.

13. The composition for use of claim 11 or 12 in inducing protective anti-C. suis immunity in sows and / or protecting their piglets against piglet coccidiosis.

14. The composition for use of any one of claims 11 to 13 in vaccinating a pregnant sow or gilt ante partum, specifically, 6 weeks, 4 weeks, and / or 2 weeks ante partum.

15. The composition for use of any one of claims 11 to 14 in re-vaccinating a sow at each weaning of its piglets.

16. The composition for use of any one of claims 11 to 15, wherein the piglets resulting from said porcine have reduced C. suis shedding and less diarrhea as compared to piglets born by an unvaccinated sow.

17. The composition for use of any one of claims 11 to 16, wherein the composition is administered as an injection, specifically an intramuscular injection.

18. A method for production of the composition of any one of claims 1 to 9, comprising the sequential steps: a. recombinant expression of HAP2, 0WP1 and TyRP proteins in bacterial host cells; b. harvesting the host cells; c. purification of each protein from the host cells; and d. mixing the proteins; e. addition of an adjuvant; and f. optionally, addition of a further protein.

19. A kit for inducing an immune response against C. suis, comprising: a. the composition according to any one of claims 1 to 9; b. instructions for use; and c. optionally, a solvent and / or an adjuvant.

Images